RNA-seq analysis of human CD8 CAR-EGFR T cells in expansion and repeated hypoxic coculture

Human CD8 EGFR CART cells during expansion and repeated hypoxic coculture were collected at different time points and subject to RNA-seq to identify key players in T cell differentiation and hypoxia-induced exhaustion. The RNA-seq data together with other functional analysis identified P4HA1 as an important enzyme that regulates T cell early differentiation and terminal exhaustion. To investigate the mechanism, P4HA1 inhibitor DPCA was used to treat the human CD8 cells during early expansion and repeated coculture and the cells were harvested for RNA-seq analysis. Overall design: Part 1) Naïve CD8 cells were isolated from PBMC of healthy donors and stimulated with CD3/CD28 and infected with CAR-virus. The CD3/CD28 antibodies and CAR virus were washed away on Day 5 and expanded in medium with IL7/15 for another 7 days before being subjected to repeated coculture under normoxia/hypoxia. CD8 CAR T cells were collected at indicated time points for RNA-seq. Part 2) Human CD8 CART cells were expanded or cocultured as described above. Cells were treated with either P4HA1 inhibitor DPCA or DMSO as vehicle control and collected at different time points for RNA-seq analysis.

本数据集的样本来源于在扩增及反复低氧共培养过程中，于不同时间点收集的人CD8 EGFR嵌合抗原受体T细胞（Chimeric Antigen Receptor T-Cell, CAR-T）。研究人员通过RNA测序（RNA-seq）分析这些样本，旨在筛选调控T细胞分化及低氧诱导耗竭的关键分子。结合RNA测序数据与其他功能实验结果，最终鉴定出P4HA1是调控T细胞早期分化及终末耗竭的关键酶类。为进一步探究P4HA1的调控机制，本研究在早期扩增及反复共培养阶段，使用P4HA1抑制剂DPCA处理人CD8 T细胞，并收集不同时间点的细胞进行RNA测序分析。

实验整体设计分为两部分：
1. 从健康志愿者的外周血单个核细胞（Peripheral Blood Mononuclear Cell, PBMC）中分离初始型（Naïve）CD8 T细胞，经CD3/CD28抗体刺激后感染CAR病毒；第5天时移除CD3/CD28抗体与CAR病毒，随后在含白细胞介素7/15（IL-7/IL-15）的培养基中继续扩增7天，之后置于常氧（normoxia）/低氧（hypoxia）环境下进行反复共培养；于指定时间点收集CD8 CAR-T细胞进行RNA测序。
2. 按照上述流程扩增并共培养人CD8 CAR-T细胞，分别使用P4HA1抑制剂DPCA或二甲基亚砜（Dimethyl Sulfoxide, DMSO，溶剂对照）处理细胞，于不同时间点收集细胞并开展RNA测序分析。